Low-level colonization of methicillin-resistant Staphylococcus aureus in pigs is maintained by slowly evolving, closely related strains in Finnish pig farms.

BACKGROUND: Over the past two decades, livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) has become widely prevalent in pig production in Europe. The carriage status of LA-MRSA is known to vary among individual pigs, but bacterial load in pigs has rarely been studied. We assessed the quantity of LA-MRSA in nasal and skin samples of pigs and investigated the genetic diversity of the strains together with sequenced strains from national surveillance and pathology samples from the Finnish Food Authority. On two farms with assumed MRSA-positive status, farm 1 and farm 2, 10 healthy pigs were sampled three times during 2 weeks from the nares and skin (study A). On farm 1, 54 additional pigs were sampled and from confirmed MRSA-positive animals, 10 were randomly selected and transported to a clean, controlled environment for further sampling (study B). From the samples taken on farms 1 and 2 and in the controlled environment, MRSA was isolated both by direct plating and enrichment on selective media. spa types, multilocus sequence types, staphylococcal cassette chromosome mec types, resistance and virulence genes were determined. Core genome multilocus sequence typing (cgMLST) analysis was performed, including the sequences deriving from the surveillance/pathology samples from the Finnish Food Authority. RESULTS: All pigs on farm 1 carried LA-MRSA in the nares at all three time points and five pigs on farm 2 at one time point. Nasal quantity varied between 10 and 103 CFU/swab and quantity on the skin between 10 and 102 CFU/swab. In the controlled environment, MRSA was detected in at least one of the nasal samples from each animal. spa type t034 was predominant. cgMLST showed one cluster with minimum allele differences between 0 and 11. CONCLUSIONS: The study shows predominantly low-level carriage (< 103 CFU/swab) of LA-MRSA on farms. In the controlled environment we observed a decline in nasal carriage but constant skin carriage. cgMLST showed that strains of spa type t034 are closely related at the national level.

Antimicrobial use, biosecurity, herd characteristics, and antimicrobial resistance in indicator Escherichia coli in ten Finnish pig farms.

We investigated connections between antimicrobial use (AMU), biosecurity, and the numbers of pigs and staff in ten Finnish farrow-to-finish herds. Data on AMU in each herd were collected for 12 months. AMU was quantified as treatment incidences per 1000 days at risk (TI) using the consensus defined daily dose calculation. Biosecurity was scored using the Biocheck.UGent™ system. We also examined antimicrobial resistance patterns of indicator E. coli isolated from faeces of selected pigs. In each herd, two groups of five pigs were formed: 1) antimicrobial treatment group (ANT: at least one pig in the litter was identified as sick and treated with antimicrobials) and 2) non-antimicrobial treatment group (NON: the litter was not medicated). Faecal samples were taken from these pigs at 5 and 22 weeks of age, cultured, and indicator E. coli isolates were tested for antimicrobial susceptibilities. The AMU varied considerably between the herds. Altogether, most of the antimicrobial treatment courses were assigned to weaned piglets. When AMU was quantified as TIs, suckling piglets had the highest TI (mean 46.6), which was significantly higher (P < 0.05) than TIs in fatteners and breeders (9.3 and 7.3, respectively). The difference between TI in suckling and TI in weaned piglets (19.1) was not statistically significant. There was a tendency for a negative correlation between the TI in breeders and the number of sows (r = -0.56, P = 0.09). Larger herds had higher external biosecurity scores than smaller herds (LS-means; 72 vs. 66, P < 0.05). The proportions of E. coli isolates resistant to at least one antimicrobial were higher in pigs at 5 weeks than in pigs at 22 weeks of age (Binomial proportion means; 40.5 % vs. 15.5 %, P < 0.05); as well as proportions of isolates resistant to at least three antimicrobial classes (23.0 % vs. 3.7 %, P < 0.01). These proportions did not differ between the ANT and NON groups at either 5 or 22 weeks of age (P> 0.05). We found few connections: enhanced external biosecurity levels found in the large herds co-occurred with lower use of antimicrobials and herds with low biosecurity scores - especially in the internal subcategories - appeared to have higher proportions of resistant isolates. Conclusively, we suggest that enhancing internal biosecurity might contribute to a reduction in the spreading of antimicrobial resistance in pig herds.

ResFinder 4.0 for predictions of phenotypes from genotypes.

OBJECTIVES: WGS-based antimicrobial susceptibility testing (AST) is as reliable as phenotypic AST for several antimicrobial/bacterial species combinations. However, routine use of WGS-based AST is hindered by the need for bioinformatics skills and knowledge of antimicrobial resistance (AMR) determinants to operate the vast majority of tools developed to date. By leveraging on ResFinder and PointFinder, two freely accessible tools that can also assist users without bioinformatics skills, we aimed at increasing their speed and providing an easily interpretable antibiogram as output. METHODS: The ResFinder code was re-written to process raw reads and use Kmer-based alignment. The existing ResFinder and PointFinder databases were revised and expanded. Additional databases were developed including a genotype-to-phenotype key associating each AMR determinant with a phenotype at the antimicrobial compound level, and species-specific panels for in silico antibiograms. ResFinder 4.0 was validated using Escherichia coli (n = 584), Salmonella spp. (n = 1081), Campylobacter jejuni (n = 239), Enterococcus faecium (n = 106), Enterococcus faecalis (n = 50) and Staphylococcus aureus (n = 163) exhibiting different AST profiles, and from different human and animal sources and geographical origins. RESULTS: Genotype-phenotype concordance was ≥95% for 46/51 and 25/32 of the antimicrobial/species combinations evaluated for Gram-negative and Gram-positive bacteria, respectively. When genotype-phenotype concordance was <95%, discrepancies were mainly linked to criteria for interpretation of phenotypic tests and suboptimal sequence quality, and not to ResFinder 4.0 performance. CONCLUSIONS: WGS-based AST using ResFinder 4.0 provides in silico antibiograms as reliable as those obtained by phenotypic AST at least for the bacterial species/antimicrobial agents of major public health relevance considered.

Host-Dependent Clustering of Campylobacter Strains From Small Mammals in Finland.

Small mammals are known to carry Campylobacter spp.; however, little is known about the genotypes and their role in human infections. We studied intestinal content from small wild mammals collected in their natural habitats in Finland in 2010-2017, and in close proximity to 40 pig or cattle farms in 2017. The animals were trapped using traditional Finnish metal snap traps. Campylobacter spp. were isolated from the intestinal content using direct plating on mCCDA. A total of 19% of the captured wild animals (n = 577) and 41% of the pooled farm samples (n = 227) were positive for C. jejuni, which was the only Campylobacter species identified. The highest prevalence occurred in yellow-necked mice (Apodemus flavicollis) and bank voles (Myodes glareolus) which carried Campylobacter spp. in 66.3 and 63.9% of the farm samples and 41.5 and 24.4% of individual animals trapped from natural habitats, respectively. Interestingly, all house mouse (Mus musculus) and shrew (Sorex spp.) samples were negative for Campylobacter spp. C. jejuni isolates (n = 145) were further characterized by whole-genome sequencing. Core genome multilocus sequence typing (cgMLST) clustering showed that mouse and vole strains were separated from the rest of the C. jejuni population (636 and 671 allelic differences, 94 and 99% of core loci, respectively). Very little or no alleles were shared with C. jejuni genomes described earlier from livestock or human isolates. FastANI results further indicated that C. jejuni strains from voles are likely to represent a new previously undescribed species or subspecies of Campylobacter. Core-genome phylogeny showed that there was no difference between isolates originating from the farm and wild captured animals. Instead, the phylogeny followed the host species-association. There was some evidence (one strain each) of livestock-associated C. jejuni occurring in a farm-caught A. flavicollis and a brown rat (Rattus norvegicus), indicating that although small mammals may not be the original reservoir of Campylobacter colonizing livestock, they may sporadically carry C. jejuni strains occurring mainly in livestock and be associated with disease in humans.

The effects of amoxicillin treatment of newborn piglets on the prevalence of hernias and abscesses, growth and ampicillin resistance of intestinal coliform bacteria in weaned pigs.

This study investigated the effects of a single amoxicillin treatment of newborn piglets on the prevalence of hernias and abscesses until the age of nine weeks. We also studied whether the treatment was associated with growth and mortality, the need for treatment of other diseases, the proportions of ampicillin resistant coliforms and antimicrobial resistance patterns of intestinal Escherichia coli (E. coli). A total of 7156 piglets, from approximately 480 litters, were divided into two treatment groups: ANT (N = 3661) and CON (N = 3495), where piglets were treated with or without a single intramuscular injection of 75 mg amoxicillin one day after birth, respectively. The umbilical and inguinal areas of weaned pigs were palpated at four and nine weeks of age. At the same time, altogether 124 pigs with hernias or abscesses and 820 non-defective pigs from three pens per batch were weighed individually. Mortality and the need to treat piglets for other diseases were recorded. Piglet faecal samples were collected from three areas of the floors of each pen at four weeks of age. The prevalence of umbilical hernias or abscesses did not differ between the groups at four weeks of age, but it was higher in the CON group than in the ANT group at nine weeks of age (2.3% vs. 0.7%, P < 0.05). Numbers of inguinal hernias and abscesses did not differ between the groups at four or nine weeks of age. The ANT group, when it compared with the CON group, increased the weight gain between four and nine weeks of age (LS means ± SE; 497.5 g/d ± 5.0 vs. 475.3 g/d ± 4.9, P < 0.01), and decreased piglet mortality (19.5% ± 1.0 vs. 6.9% ± 1.0, P < 0.05) and the need to treat the piglets for leg problems (3.4% ± 0.3 vs. 1.9% ± 0.3%, P < 0.01) but not for other diseases by the age of four weeks. The proportion of ampicillin resistant intestinal coliform bacteria and the resistance patterns of the E. coli isolates were not different between the ANT and CON groups. In conclusion, our results showed that the amoxicillin treatment of new-born piglets produced statistically significant effect in some of the parameters studied. However, as these effects were only minor, we did not find grounds to recommend preventive antibiotic treatment. Further, continuous antimicrobial treatment of newborn piglets could negatively influence the development of the normal microbiota of the piglet and promote selection of antimicrobial resistance genes in herds. Therefore we suggest rejection of the use of routine administration of antimicrobial agents at birth.

Functional genomics in Campylobacter coli identified a novel streptomycin resistance gene located in a hypervariable genomic region.

Numerous aminoglycoside resistance genes have been reported in Campylobacter spp. often resembling those from Gram-positive bacterial species and located in transferable genetic elements with other resistance genes. We discovered a new streptomycin (STR) resistance gene in Campylobactercoli showing 27-34 % amino acid identity to aminoglycoside 6-nucleotidyl-transferases described previously in Campylobacter. STR resistance was verified by gene expression and insertional inactivation. This ant-like gene differs from the previously described aminoglycoside resistance genes in Campylobacter spp. in several aspects. It does not appear to originate from Gram-positive bacteria and is located in a region corresponding to a previously described hypervariable region 14 of C. jejuni with no other known resistance genes detected in close proximity. Finally, it does not belong to a multiple drug resistance plasmid or transposon. This novel ant-like gene appears widely spread among C. coli as it is found in strains originating both from Europe and the United States and from several, apparently unrelated, hosts and environmental sources. The closest homologue (60 % amino acid identity) was found in certain C. jejuni and C. coli strains in a similar genomic location, but an association with STR resistance was not detected. Based on the findings presented here, we hypothesize that Campylobacter ant-like gene A has originated from a common ancestral proto-resistance element in Campylobacter spp., possibly encoding a protein with a different function. In conclusion, whole genome sequencing allowed us to fill in a knowledge gap concerning STR resistance in C. coli by revealing a novel STR resistance gene possibly inherent to Campylobacter.

Population Genetics and Antimicrobial Susceptibility of Canine Campylobacter Isolates Collected before and after a Raw Feeding Experiment.

In recent years, increasing numbers of consumers have become interested in feeding raw food for their pet dogs as opposed to commercial dry food, in the belief of health advantages. However, raw meat and internal organs, possibly contaminated by pathogens such as Campylobacter spp., may pose a risk of transmission of zoonoses to the pet owners. Campylobacter jejuni is the leading cause of bacterial gastroenteritis in humans but C. upsaliensis has also been associated with human disease. In this study we investigated the effect of different feeding strategies on the prevalence of Campylobacter spp. in Finnish dogs. We further characterized the isolates using multilocus sequence typing (MLST), whole-genome (wg) MLST and antimicrobial susceptibility testing. Dogs were sampled before and after a feeding period consisting of commercial raw feed or dry pellet feed. Altogether 56% (20/36) of the dogs yielded at least one Campylobacter-positive fecal sample. C. upsaliensis was the major species detected from 39% of the dogs before and 30% after the feeding period. Two C. jejuni isolates were recovered, both from raw-fed dogs after the dietary regimen. The isolates represented the same genotype (ST-1326), suggesting a common infection source. However, no statistically significant correlation was found between the feeding strategies and Campylobacter spp. carriage. The global genealogy of MLST types of dog and human C. upsaliensis isolates revealed weakly clonal population structure as most STs were widely dispersed. Major antimicrobial resistance among C. upsaliensis isolates was against streptomycin (STR MIC > 4 mg/l). Apart from that, all isolates were highly susceptible against the antimicrobials tested. Mutations were found in the genes rpsL or rpsL and rsmG in streptomycin resistant isolates. In conclusion, increasing trend to feed dogs with raw meat warrants more studies to evaluate the risk associated with raw feeding of pets in transmission of zoonoses to humans.

Longitudinal on-farm study of the development of antimicrobial resistance in Campylobacter coli from pigs before and after danofloxacin and tylosin treatments.

Effects of danofloxacin or consecutive fluoroquinolone and macrolide treatments on resistance development in Campylobacter have remained uncharacterised. Therefore we analysed the development of resistance in porcine Campylobacter coli before and after danofloxacin and tylosin treatments at a farrowing farm. Danofloxacin-treated (n=12, group A) and control pigs (n=15, group B) were subsequently treated with tylosin and sampled longitudinally. C. coli were isolated and susceptibilities to ciprofloxacin and erythromycin were assessed, isolates were genotyped with PFGE and resistance-related mutations were identified. Isolates from the danofloxacin-treated pigs had more frequently non-wild type MICs (above the epidemiological cut-off value (ECOFF)) for ciprofloxacin (P<0.001) and erythromycin (P<0.05) than those isolated before danofloxacin or those from the controls. Subsequent tylosin treatment increased proportion of isolates with non-wild type MICs for erythromycin in both groups A and B (P<0.01) and, interestingly, proportion of isolates with non-wild type MICs for ciprofloxacin in group B (P<0.001) with high MICs (128 µg/ml). PFGE analysis revealed treatments selecting predominant genotypes with variable resistance patterns and decreasing initial diversity of genotypes. The most common genotype had mainly high MICs for ciprofloxacin among danofloxacin-treated pigs but wild type MICs (below the ECOFF) among the controls housed in the same pens. This suggests that the non-wild type isolate was rarely transmitted or outcompeting wild type genotype in the control pigs without selection pressure. Isolates exhibiting non-wild type MICs for ciprofloxacin harboured the C257T (Thr-86-Ile) mutation in the gyrA gene. In conclusion, a high dose of danofloxacin used at the farm did not prevent emergence of isolates with high MICs for ciprofloxacin. After subsequent tylosin treatment isolates had even higher MICs for ciprofloxacin and erythromycin than before the treatment. Therefore, controlled use of antimicrobials in food animal production is essential.

Mutations in the rpsL gene are involved in streptomycin resistance in Campylobacter coli.

To characterize the mechanisms of streptomycin (STR) resistance in Campylobacter coli, we chose 17 isolates that were resistant to STR, erythromycin (ERY), or both, and the putative STR resistance target genes rpsL, rrs, and gidB were analyzed for mutations. The presence of the aadE gene encoding aminoglycoside 6-adenylyltransferase was also evaluated. To reveal putative connection between ERY and STR resistance mechanisms, 13 C. coli isolates initially susceptible to STR and ERY were exposed to STR, and resistant variants were characterized. We also assessed the development of ERY resistance with a similar method. Finally, the effect of the putative CmeABC efflux pump inhibitor phenyl-arginine-beta-naphthylamine on STR resistance was tested. Our studies showed an association between mutations in the rpsL gene and STR resistance in C. coli. Further, mutations obtained in vitro were more diverse than those occurring in vivo. However, we observed no resistance associated mutations in the other genes studied, and selection with STR did not result in variants resistant to ERY and vice versa. None of the isolates harbored the aadE gene, and no differences in STR minimum inhibitory concentration levels were detected in the presence or absence of phenyl-arginine-beta-naphthylamine. In conclusion, we found that STR resistance was associated with mutations in the rpsL gene, but no obvious association between STR and ERY resistance mechanisms was found in C. coli.